Abstract

A major challenge in the study of proteomics is the analysis of complex samples. As sample complexity increases so does the need for a highly sensitive instrument with large dynamic range capabilities. Also required for deeper analysis of complex samples is the ability to perform high speed MS/MS scans without loss of spectral quality during an LC-MS/MS run. In the current study using a QqTOF instrument, we used a new feature that enables dynamic acquisition time of MS/MS spectra based on the evaluation of fragment ion intensity in combination with repetitive LC-MS/MS runs that exclude previously acquired precursors. Utilizing this intelligent MS/MS acquisition feature we achieved low attomole level sensitivity and four-log orders of dynamic range on a commercially available sample. Analysis of more complex samples (i.e. animal tissue) resulted in the identification of hundreds of nonredundant proteins from single cation exchange fractions with greater than 95% confidence using 45 minute gradients. The use of dynamic acquisition time and the repetitive LC strategy combined with ProteinPilot software illustrate the effectiveness of our approach in identifying a large number of nonredundant proteins with high confidence in a relatively short time.

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